SNP molecular marker of sheep ERCC6 gene and its application

Abstract

The invention discloses a sheep ERCC6 gene SNP molecular marker and its application, belonging to the field of molecular biology, detecting the genotype of the 43219309 position of chromosome 25 of sheep, the genome version Oar_v3.1, GCF_000298735.1, the base of the site has G / A polymorphism, and when the genotype of the site is GA, it has higher growth performance than GG. It can be used as a molecular marker of sheep growth traits.

Description

Technical Field

[0001] The invention belongs to the field of molecular biology, and specifically relates to a sheep ERCC6 gene SNP molecular marker and application thereof in sheep breeding. Background Art

[0002] China is a large livestock breeding country. Improving the growth performance of sheep is an important goal in the sheep breeding process, which can bring huge economic benefits to the sheep industry. The growth performance of sheep is mainly related to the growth and development of sheep. Sheep with fast growth rate and larger body have relatively excellent growth performance. In the sheep genome, major effect genes or molecular marker sites related to growth and development can be applied to meat sheep breeding, but there are currently few molecular markers suitable for breeding sheep with excellent growth performance. Mining molecular markers related to sheep growth can provide targets for sheep molecular marker-assisted selection breeding.

[0003] ERCC6 (Excision repair cross-complementing group 6, ERCC6) is an important DNA repair protein that plays a role in transcription-coupled excision repair, DNA repair process (Chang et al., 2009; Takahashi et al., 2019) and cell division (Paccosi et al. 2020). This protein has ATPase activity and interacts with transcription and excision repair proteins to promote the formation of DNA repair site complexes. ERCC6 is often upregulated in cancer cells, which is conducive to cancer cell proliferation and inhibits cell apoptosis. It has been recognized as an important target for cancer treatment (Proietti-De-Santis et al., 2018). Zou et al. found that ERCC6 can reduce apoptosis, inflammation, aging and oxidative stress in spinal cord injury mice and cell models (Zou et al., 2022), indicating that this gene is closely related to animal growth and development. However, the direct effect on sheep growth and development and its mechanism of action have not been reported. Summary of the invention

[0004] The technical problem to be solved by the present invention is to provide a SNP molecular marker related to sheep growth traits and to provide a new molecular marker site for sheep growth trait determination.

[0005] The technical solution of the present invention is: a method for breeding sheep with high growth performance, detecting the genotype of position 43219309 of chromosome 25 of sheep, the genome version is Oar_v3.1, GCF_000298735.1, the base of the site has G / A polymorphism, and when the genotype of the site is GA, it has higher growth performance than GG.

[0006] The invention discloses an application of a material for detecting the genotype of a SNP site in sheep growth performance assisted breeding, wherein the SNP site is located at position 43219309 of chromosome 25 of sheep, located at position 170 of the nucleotide sequence described in SEQ ID No. 1, and has a G / A polymorphism.

[0007] Furthermore, the substance is a primer pair, and the nucleotide sequence of the primer pair is shown in SEQ ID No.2 and SEQ ID No.3.

[0008] Compared with the prior art, the present invention has the following beneficial effects:

[0009] By using the sheep 40K chip to perform genotyping on 567 Hu sheep individuals with weight records, combined with known pedigree information and phenotypic information, statistical methods were used to conduct association analysis on growth phenotypic indicators such as weight at different growth stages, and it was found that a SNP on the ERCC6 gene was associated with the sheep growth phenotype. The SNP was located at position 43219309 on chromosome 25 of the sheep (genome version Oar_v3.1, GCF_000298735.1), and had a G / A polymorphism. When the genotype of the SNP site mutated to GA, it had higher growth performance than the wild type GG, and could be used as a molecular marker for sheep growth traits. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Figure 1 It is the gel electrophoresis diagram of the PCR amplification product containing the target SNP site;

[0011] Figure 2 The sanger sequencing results of the PCR amplified product fragment;

[0012] Figure 3 Comparing the body weights of Hu sheep individuals containing this molecular marker (GA, heterozygous mutant) and Hu sheep individuals not containing this molecular marker (GG, homozygous wild type) at different ages, the body weights of mutant individuals at different ages were all higher than those of wild-type individuals. DETAILED DESCRIPTION

[0013] The experimental methods in the following examples are conventional methods unless otherwise specified. The experimental materials used in the following examples are purchased from commercial channels unless otherwise specified.

[0014] Example 1 Discovery of ERCC6 gene SNP molecular markers

[0015] By using the sheep 40K chip to perform genotyping on 567 Hu sheep individuals with weight records, combined with known pedigree information and phenotypic information, statistical methods were used to conduct association analysis on growth phenotypic indicators such as weight at different growth stages, and it was found that a SNP on the ERCC6 gene was associated with the sheep growth phenotype. The SNP was located at position 43219309 on chromosome 25 of the sheep (genome version Oar_v3.1, GCF_000298735.1), and had a G / A polymorphism. When the genotype of the SNP site mutated to GA, it had higher growth performance than the wild type GG, and could be used as a molecular marker for sheep growth traits.

[0016] Example 2 Detection of SNP Molecular Markers

[0017] (1) The SNP is located at position 170 of the nucleotide sequence shown in SEQ ID No. 1. Based on the upstream and downstream sequences of the SNP site, specific primers for amplifying the fragment were designed:

[0018] Upstream primer ERCC6-F1: 5′-AGATGAGACACAGCCAGGAT-3′

[0019] Downstream primer ERCC6-R1: 5′-ATTGGGCAGCTGTTAACTCG-3′

[0020] (2) PCR amplification was performed on the sheep DNA sample using the specific primers of (1) to obtain an amplification product of the region containing the mutation. The amplification product was 531 bp ( Figure 1 ).

[0021] The amplification system is as follows:

[0022] Table 1JAK2 amplification system

[0023]

[0024] Max Master Mix is ​​a high-fidelity PCR enzyme from Nanjing Novozymes Biotech Co., Ltd., catalog number P525-02.

[0025] The amplification conditions were as follows: amplification was performed using a PCR amplification instrument, the first stage: pre-denaturation at 95°C for 3 min; the second stage: denaturation at 95°C for 30 s, annealing at 59°C for 30 s, extension at 72°C for 30 s, 35 cycles; the third stage: final extension at 72°C for 5 min, and storage at 4°C.

[0026] (3) The amplified product was subjected to Sanger sequencing ( Figure 2 ) to obtain the genotype of the SNP site.

[0027] Example 3 Verification of SNP Molecular Markers

[0028] The genomes of 242 sheep (the breed was Hu sheep, and the weights at different growth stages were shown in Table 2) were amplified. The amplification primers, conditions, and system were the same as those in Example 2. The genotypes of the PCR products after sequencing are shown in Table 2.

[0029] Table 2 Body weight and genotype of 242 sheep

[0030]

[0031]

[0032]

[0033]

[0034]

[0035]

[0036] The results are shown in Table 2. 51 sheep were heterozygous individuals (GA) containing the SNP site mutation, 191 were wild-type individuals (GG, genotype consistent with the reference genome), and no homozygous mutant individuals with genotype AA were found. Therefore, the growth performance of homozygous mutant individuals could not be determined. The birth weight and weight at different ages of all individuals were compared, and the growth curve was drawn with the mean ± standard deviation of the weight of individuals with the same genotype ( Figure 3 ), it can be seen that the heterozygous mutant individuals containing this molecular marker have higher body weight at different ages than wild-type individuals.

Claims

1. A method for breeding sheep with high growth performance, characterized in that: The genotype of position 43219309 of chromosome 25 of sheep was detected, the genome version was Oar_v3.1, GCF_000298735.1, the base of this position had G / A polymorphism, when the genotype of this position was GA, it had higher growth performance than GG, the growth performance was body weight.

2. Application of a material for detecting the genotype of a SNP site in sheep growth performance assisted breeding, wherein the SNP site is located at position 43219309 of chromosome 25 of sheep, genome version Oar_v3.1, GCF_000298735.1, located at position 170 of the nucleotide sequence described in SEQ ID No. 1, and has a G / A polymorphism. When the genotype of the site is GA, it has a higher growth performance than GG, and the growth performance is body weight.

3. The use according to claim 2, characterized in that: The substance is a primer pair, and the nucleotide sequence of the primer pair is shown as SEQ ID No.2 and SEQ ID No.3.

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